Rapid single B cell antibody discovery using nanopens and structured light
Rapid single B cell antibody discovery using nanopens and structured light
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DOI：
10.1080/19420862.2019.1624126
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发表时间：
2019-06-13
期刊：
影响因子：
5.3
通讯作者：
King, Chadwick T.
中科院分区：
文献类型：
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作者：
Winters, Aaron;McFadden, Karyn;King, Chadwick T.
在当今快节奏和竞争激烈的药物开发市场中，单克隆抗体（mAb）工具试剂的加速开发是成功推进治疗性抗体的基本要求。在这里，我们描述了一种直接、灵活、快速的纳米流体光电单B淋巴细胞抗体筛选技术（NanOBlast），应用于抗独特型试剂抗体的产生。从脾脏和淋巴结中收获选择性富集、抗原经历的小鼠抗体分泌细胞（ASCs）。随后，使用一种新型的、集成的、高含量的培养和检测平台直接筛选从单独分离的单个ASCs中分泌的单克隆抗体，该平台能够使用结构光操纵微流控芯片纳米孔中的活细胞。通过单细胞聚合酶链反应对抗独特型ASCs进行分子回收，然后进行重组IgG表达和酶联免疫吸附试验（ELISA）鉴定，获得了多种高亲和力的抗独特型试剂单克隆抗体。组合ELISA筛选确定了捕获和检测单克隆抗体，并开发了一种敏感且高度特异性的配体结合试验，能够直接从人类患者血清中定量游离治疗性IgG分子，从而促进了重要的药物开发决策。芯片上ASC的导入、筛选和导出发现流程在5 h内完成，而从免疫到重组表达IgG的整个发现流程在60天内完成。
Accelerated development of monoclonal antibody (mAb) tool reagents is an essential requirement for the successful advancement of therapeutic antibodies in today's fast-paced and competitive drug development marketplace. Here, we describe a direct, flexible, and rapid nanofluidic optoelectronic single B lymphocyte antibody screening technique (NanOBlast) applied to the generation of anti-idiotypic reagent antibodies. Selectively enriched, antigen-experienced murine antibody secreting cells (ASCs) were harvested from spleen and lymph nodes. Subsequently, secreted mAbs from individually isolated, single ASCs were screened directly using a novel, integrated, high-content culture, and assay platform capable of manipulating living cells within microfluidic chip nanopens using structured light. Single-cell polymerase chain reaction-based molecular recovery on select anti-idiotypic ASCs followed by recombinant IgG expression and enzyme-linked immunosorbent assay (ELISA) characterization resulted in the recovery and identification of a diverse and high-affinity panel of anti-idiotypic reagent mAbs. Combinatorial ELISA screening identified both capture and detection mAbs, and enabled the development of a sensitive and highly specific ligand binding assay capable of quantifying free therapeutic IgG molecules directly from human patient serum, thereby facilitating important drug development decision-making. The ASC import, screening, and export discovery workflow on the chip was completed within 5 h, while the overall discovery workflow from immunization to recombinantly expressed IgG was completed in under 60 days.